The goal of the proposed research is to characterize at the molecular level how mutations can alter the substrate profile of the clinically important TEM-1 beta-lactamase. These specific questions will be addressed: 1. What beta-lactamase mutations cause an increase in resistance to beta-lactam antibiotics? Every amino acid position in TEM-1 beta-lactamase will be randomized to sample all possible amino acid substitutions using a novel mutagenesis technique. All of the random substitutions will then be screened to identify those amino acid substitutions that result in increased beta-lactamase activity towards a set of clinically relevant beta-lactam antibiotics. 2. How do mutations change the substrate spectrum of beta-lactamase? Biochemical methods will be used to further characterize the new hydrolytic capacities of the specificity mutants. These studies will include purification of the mutant proteins and determination of kinetic parameters. The conformational stability of the mutant proteins also will be determined by a variety of methods. 3. What beta-lactamase mutations result in increase antibiotic resistance in clinical isolates? The information obtained from the above studies will be used to design oligonucleotide probes that will specifically detect genes encoding specificity mutants among TEM beta-lactamase-containing clinical isolates. The studies will test the predictive capacity of the random substitution experiments and provide specific information about the mechanisms of beta-lactam antibiotic resistance. In total, these studies will assess systematically the ability of TEM-1 beta-lactamase to expand its substrate spectrum by mutation. The information gained in these studies will be of immediate empirical value in predicting the susceptibility of a given antibiotic to mutational changes in the beta-lactamase. In the long term we would like to use the data to predict how beta-lactamase will respond to the selective pressure of antibiotic therapy.